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Fig. 21.3 MTOC positioning at the immunological synapse. CTLs were activated by EL4.BU
target cells and immunostained with tubulin (a) or LFA-1 (red) and tubulin (green)(b, c, d). The
target cells were treated with colchicine to depolymerize microtubules prior to pairing for clarity.
Three-dimensional reconstructions were derived from the fluorescence images. a In the late stage of
MTOC translocation, microtubules appear to contact the pSMAC and then bend backwards away
from the target cell. b The MTOC can be seen in the middle of the target contact site defined by the
ring of LFA-1. c, d Cropped and rotated views of the synapse. In c, the MTOC is centered in the LFA-
1 ring. In d, the MTOC is located toward the top of the LFA-1 ring image. No images obtained during
these experiments showed the MTOC outside of the LFA-1 ring. Figures a-d are reprinted from
Immunity 16:1 Kuhn and Poenie Dynamic polarization of the microtubule cytoskeleton during
CTL-mediated killing. Copyright (2002) with permission from Elsevier
b
LFA-1 rings, the MTOC generally localized close to the partial ring, as opposed to
activated T cells with full rings, where the MTOC was generally found close to the
center of the ring.
The imaging data suggested that points where microtubules contact the cortex
formed a ring-like pattern that correlated with the pSMAC and suggested the
hypothesis that dynein might be organized similarly. To further investigate the role of
dynein in MTOC translocation, we used Jurkat T cells. These cells have a number of
advantages and disadvantages when compared to the mouse CTL line we used for
MPM studies. The main disadvantage is that they are a T helper tumor line and less
representative of normal T cells. Furthermore, the Jurkat TcR-antigen specificity is
not known. As a substitute for an antigen-presenting target cell, we used Raji cells (a
B cell line) coated with Staphylococcus enterotoxin E (SEE), a superantigen. The
superantigen mimics the normal TcR-peptide-MHC interaction insofar as it binds to
both the Jurkat TcR and the Raji MHC (Muller-Alouf et al.
2001
).
The strong Jurkat cell TcR engagement by SEE, while an artificial stimulus, also
potentially has certain advantages when it comes to trying to understand the basic
mechanism of MTOC translocation. Signal transduction through the TcR is complex
and a number of molecules may be important for T cell activation under conditions of
weak TcR-antigen binding that might not otherwise be necessary. There are a number
of molecules involved in T cell adhesion and signaling that not only affect MTOC
reorientation but also impact numerous signaling events. Examples include the
integrin LFA-1 (lymphocyte function-associated antigen-1) (Davignon et al.
1981
;
Li et al.
2009
; Davignon et al.
1981
; Anikeeva et al.
2005
), Fyn kinase (Martin-
Cofreces et al.
2006
), and Vav, a guanine nucleotide exchange factor for small G
proteins (Ardouin et al.
2003
). When CTLs are treated with monoclonal antibodies to
LFA-1, their cytolytic function is profoundly inhibited (90 %) (Davignon et al.
1981
). On the other hand, when LFA-1-deficient Jurkat cells stimulated by SEE-
coated Raji cells, MTOC reorientation was not greatly affected (Combs et al.
2006
).
Similarly, siRNA knockdown of Fyn to undetectable levels in Jurkat cells also had
little effect on MTOC translocation (Tan and Poenie, unpublished observations).
With respect to Vav, Ardouin and colleagues showed that for Vav
-/-
thymocytes,
MTOC reorientation was reduced from 74 % to 49 %. This partial reduction of
MTOC translocation correlates with impaired calcium elevation and signaling
events. It was noted, however, that in Vav-1
-/-
Jurkat cells, these effects were less
